The project was initiated to identify and characterize tumor-associated protein changes during both human and experimental mammary carcinogenesis. Analysis of polypeptide changes during human mammary carcinogenesis revealed both qualitative and quantitative polypeptide differences. Six cytosolic polypeptides were expressed in all malignant tissues (eight individuals) but not in normal tissue, while one polypeptide (p52; pI 7.40/52 kDa) was not expressed during carcinogenesis. More numerous quantitative changes were also noted. These changes were localized mainly in the pI range 5.8-7.0 and molecular weight ranges of 22-40 kDa. Comparison of polypeptides in this region revealed a general up-regulation of polypeptide expression in malignant tissues as compared to those from normal mammary tissue. Included in this group of polypeptides is one polypeptide, p24 (pI 6.15/24 kDa), which was expressed in greatest concentrations in tissues exhibiting the highest estrogen receptor (ER) content. Expression of p24 was markedly reduced in normal tissue and in malignant tumors possessing low levels of progesterone receptor (PgR)/ER content. This polypeptide (p24) is distinct from any of the major known milk proteins and from the 24K protein of MCF-7 cells, a well-studied differentiation marker in human breast cancer.